1. Field of the Invention
The present invention relates to an assay for the analysis and selection of potatoes having a high content of Proteinase Inhibitor II (PI2), and more specifically, to a fast and inexpensive assay of PI2 content of whole potatoes to be used as raw material in a process for extracting PI2 from the whole potatoes.
2. Background of the Prior Art
Proteins that inhibit proteolytic enzymes are often found in high concentrations in many seeds and other plant storage organs. Inhibitor proteins are also found in virtually all animal tissues and fluids. These proteins have been the object of considerable research for many years because of their ability to complex with and inhibit proteolytic enzymes from animals and microorganisms. The inhibitors have become valuable tools for the study of proteolysis in medicine and biology. Proteinase inhibitors are of particular interest due to their therapeutic potentials in controlling proteinases involved in a number of disorders such as pancreatitis, shock, and emphysema, and as agents for the regulation of mammalian fertilization. Potato tubers are a rich source of a complex group of proteins and polypeptides at potently inhibit several proteolytic enzymes usually found in animals and microorganisms. In particular, potato inhibitors are known to inhibit human digestive proteinases, and thus have application in the control of obesity and diabetes.
Proteinase inhibitors extracted from potatoes have been distinguished into two groups based on their heat stability. The group of inhibitors that is stable at 80° C. for 10 minutes have been identified as inhibitor I (mol. wt. 39,000) (Melville, J. C. and Ryan, C. A. Chymotrypsin inhibitor I from potatoes. J. Biological Chem., 247: 3445-3453, 1972), carboxypeptidase inhibitor (CPI) (mol. wt. 4,100) (Ryan, C. L., Purification and properties of a carboxypeptidase inhibitor from potatoes. J. Biol. Chem. 249: 5495-5499, 1974), inhibitors IIa and IIb (mol. wt. 20,700) (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976), and inhibitor A5 (mol. wt. 26,000).
In 1972, Melville and Ryan (Melville et al.) reported a large-scale preparation for isolating Chymotrypsin Inhibitor I from potato tubers. According to the method of Melville and Ryan, potatoes were sliced with peels intact and soaked in a sodium dithionite solution, homogenized, and expressed through nylon cloth. The resulting juice was adjusted to a pH of 3, centrifuged at 1000×g for 15 minutes at 5° F. and the supernatant collected and fractionated with ammonium sulfate.
Purification was achieved through water washing and heat treatment whereby clear filtrates of heated fractions were pooled and lyophilized. Suspending the lyophilized powder in water, dialyzing it against water for 48 hours, and lyophilizing the resulting clear filtrate obtained a crude extract. Resuspended extract was then centrifuged and applied to a column of Sephadex G-75. Collected fractions containing the Inhibitor I were pooled, evaporated, and desalted on a column of Sephadex G-25. The resulting gel-filtered inhibitor product was determined to be approximately 90% Inhibitor I protein purified by dissociation on a Sephadex G-75 column and desalted on a column of Sephadex G-25.
The Ryan lab followed-up by reporting the isolation and characterization of Proteinase Inhibitor II in much the same manner as described for Inhibitor I (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976). Bryant et al. differentiated potato-derived proteinase inhibitors into two groups based on their respective stabilities to a temperature of 80° C. for 10 minutes. Proteinase Inhibitor I (PI1) is characterized as a tetrameric protein composed of four hybridized isoinhibitor protomer species having a molecular weight of 39,000, whereas PI2 is characterized as a dimeric inhibitor comprising four isoinhibitor promoter species having a molecular weight of 21,000.
The isolation of proteinase inhibitor proteins from potatoes is described in WO 99/01474. Proteins from potato tubers are extracted in soluble form in an aqueous/alcohol extraction medium, such as dilute formic acid and 20% ethanol. The alcohol extract is heated to a first temperature to denature most of the unwanted proteins and cooled to a second temperature to form a precipitate phase constituting the debris and a soluble phase that contains the heat stable proteinase inhibitor proteins. The heat stable proteinase inhibitor proteins are precipitated from the soluble phase by dialysis against a suitable dialysis medium, such as dilute formic acid.
The production of PI2 on a commercial scale will be benefited by the identification and use of whole potatoes as starting material that have a high level of PI2. Such raw material will increase the amount of PI2 obtained from a given amount of starting material, will increase the efficiency of the extraction and purification steps, will reduce the amount of waste material, and will lower the cost of the PI2 product. Additionally, the raw material chosen is preferably be readily available in production-scale (truckload) quantity, and contains PI2 in consistent concentration.
Researchers have previously characterized a number of varieties of potatoes according to their proteinase inhibitor content (Ryan, C. A.; Kuo, T.; Pearce, G.; Kunkel, R. Variability in the concentration of three heat stable proteinase inhibitor proteins in potato tubers. American Potato Journal, 53: 443-454. 1976). In the study, PI2 was purified using chromatography, as described in Bryant, et al, supra. In summary, the method uses radial diffusion quantitation which requires radial assay materials, including spotting media and standards. Antibodies developed from antibody serum used in the assay had to be independently developed in the researcher's laboratory. The assay requires almost three months to generate useful data. The paper reports that there was a positive correlation (a correlation coefficient of 0.70) of both PI1 and PI2 with total soluble protein and proposed that the proteinase inhibitors could be used as excellent markers for genetic studies for selecting high protein potato tuber varieties.
The known assay for PI2 is time-consuming, labor intensive, was demonstrated only at laboratory scales, and expensive. Selection criteria currently evaluated by the potato processing industry have included total starch, sugar, water, and protein contents, but not PI2 content. Contrary to the conclusion reported in the American Potato Journal, the present assay has shown that there is no substantial correlation between total protein content and PI2 content. There is a need for an assay for PI2 that is rapid and inexpensive, and which will accurately forecast the yield of PI2 when the tested raw materials are used in a commercial PI2 extraction facility.